This invention relates to a multipurpose reagent system and a method for a rapid analysis of whole blood samples. More particularly, the present invention relates to a multipurpose reagent system capable of rapidly lysing red cells and concurrently fixing white cells, useful for performing white cell differential analyses and quantitative analyses of nucleated red blood cells or lymphocyte subclassification using immunophenotyping techniques on an automated clinical hematology analyzer or flow cytometer.
The peripheral blood of a normal subject contains red blood cells, also known as erythrocytes, and five major classes of mature white cells, also known as leukocytes. There are at least five classes of leukocytes, known as neutrophils, eosinophils, monocytes, lymphocytes and basophils. Each type of mature blood cell performs specialized functions necessary in maintaining the homeostasis of the host. The concentration of each class of peripheral blood cells is tightly regulated and monitored by a dynamic process involving a variety of factors present in the microenvironment of the bone marrow. Under certain disease conditions, the bone marrow may release either an increased or decreased number of certain classes of white cells. In other conditions, all regulation of she number of peripheral blood cells released from the bone marrow is perturbed and an uncontrolled number of immature white or red cells are released to the peripheral blood.
Therefore, monitoring the concentration of the five normal classes of leukocytes and identifying the presence of immature erythrocytes and leukocytes in the peripheral blood is an important diagnostic tool for physicians. Typically, these functions have been performed by doing white cell differential counts, whereby the relative proportions of the five normal classes of leukocytes and any abnormal cells are determined microscopically. The manual procedure is very time consuming, subjective and labor intensive.
Recently, automated processes and automated flow system apparatuses have been developed to ease the burden of white cell differential analysis. Several of these systems are described in U.S. Pat. Nos. 4,099,917; 4,617,275; 4,521,518; and 4,801,549. Some of these systems are based on cytochemical procedures to specifically identify individual cell types; some of these systems differentiate three leukocyte types by electronic impedance measurements of cell volume; and other procedures utilize a combination of optical and electronic impedance measurements to differentiate the five classes of peripheral white blood cells.
Recent advances in cellular immunology and flow cytometry are being utilized to identify and quantify lymphocyte subclasses such as helper T cells. Lymphocyte subclassification has become an important diagnostic tool, particularly in view of the growing AIDS epidemic. Conventional lymphocyte subclassification involves the following steps: (1) The separation of lymphocytes from other peripheral blood cells by density gradient centrifugation; (2) the reaction of the lymphocytes with fluorochrome-labeled monoclonal antibodies directed to specific lymphocyte surface antigens; and (3) the analysis of lymphocyte-antibody reaction products using flow cytometry. Currently, a great deal of effort is being directed towards the development of whole blood methods that bypass the need for density gradient centrifugation. Recently developed whole blood methods for lymphocyte subclassification comprises lysing the red cells, removing red cell ghosts and cell debris by centrifugation, and preserving the morphology of the remaining white cells by suspending the white cells in an isotonic saline solution containing appropriate fixatives. Although these methodologies avoid the need for density gradient centrifugation, they are still incompatible with available automated clinical hematologic analyzers since they still require a centrifugation step.
Generally speaking, the reagent systems available for use during the analysis of nucleated red blood cells (NRBC) are as yet unable to allow for the differentiation and counting of NRBC signals from red cell stroma or large platelets and only allow the instrument to flag possible NRBC signals.
It is imperative in leukocyte analyses that all of the red blood cells be completely lysed. Since red cells outnumber white cells by about 700 to 1, even one percent of unlysed red cells may distort white cell counts. Some reagents used to lyse red cells require too lengthy an incubation period to be practical in an automated clinical analyzer. For example, the Tris buffered ammonium chloride solution recommended by K. A. Murihead in Clinical Cytometry, Ann. N.Y. Acd. Sci., vol. 468, pp. 113-127 (1986) takes 5 to 10 minutes to lyse red cells, which is too impractical for automation.
Furthermore, incomplete hemolysis with certain lytic reagents can result in red cell stroma that retain sufficient hemoglobin to generate high background counts in automated clinical electro-optical systems. Therefore, the white cells to be analyzed must first be removed from the red cell stroma by centrifugation, a procedure that is a limiting factor when adapting a reagent system for automation.
Other reagent systems, such as those described in U.S. Pat. Nos. 4,902,613 and 4,654,312, that are used to lyse red cells, contain high refractive index solvents. A cell suspending medium which has a high refractive index has two disadvantages: (1) The refractive index may be too high for a common flow cell saline sheath; and (2) the high refractive index of the suspending medium may mask signals from small cellular components such as small lymphocytes and cytoplasm-lysed nucleated red cells. Thus, before the cells can be analyzed in a flow cell, the cells must be removed from the high refractive index medium by centrifugation and resuspended in an isotonic solution. Such manual procedures are not desirable or adaptable for use on a fully automated clinical analyzer.
In addition, lytic reagents, such as those described in U.S. Pat. No. 5,155,044, are too hypotonic and/or acidic. Such lysing reagents require the rapid "follow-up" addition of a high salt solution and/or alkaline salt solution to preserve the white cell morphology for analysis. Similarly, lyric reagents, such as those described in U.S. Pat. No. 4,751,179, will not only lyse red cells but will also lyse white cells, unless a separate fixative is added at the appropriate time and concentration to prevent white cell lysis. These reagents introduce the potential of white cell damage, particularly in abnormal blood samples containing fragile white cells (such as in blood samples from patients with chronic lymphocytic leukemia [CCL]).
Furthermore, reagent systems, such as those described in U.S. Pat. Nos. 4,099,917, 4,801,549, and 4,978,624, require incubations at high temperatures, e.g. over 50.degree. C., to completely lyse the red cells. Temperatures over 45.degree. C. will, generally, begin to denature most cell surface antigens and cause hemoglobin clumping in the process. Although these systems may be used to perform differential analyses of white cells, they destroy the means for differentiating subpopulations of lymphocytes and cannot be used for immunophenotypic lymphocyte classification.
Many of the currently used reagent systems require the cytochemical staining of fixed white cells before they are subjected to differential analysis. These systems require the timed addition of multiple reagents and incubation periods and are generally not adaptable for the quantitation of nucleated red cells or for immunophenotypic lymphocyte classification. Furthermore, each step of reagent addition or other manipulation of a blood sample decreases the precision of the final counts obtained from that sample.
Based on the foregoing, a need has arisen for a multipurpose reagent system which can lyse red cells rapidly and completely, while concurrently preserving white cell morphology and lymphocyte cell surface antigens.